Translocation of the HIV-1 genome into the nucleus of a target cell is a critical step in viral replication in both dividing and non-dividing cells. This process can be attributed to the interaction between the viral preintegration complex and the cellular nuclear import machinery. One of the main regulators of this interaction is viral protein R (Vpr). However, molecular details of Vpr's activity are not understood. In addition, nuclear import of HIV in some cell types appears to be Vpr-independent. Our recent studies have identified heat shock protein 70 (Hsp70) as a substitute for Vpr in such cells, but the molecular reason for this substitution is not known. Our long-range goal is to identify novel targets for anti-HIV intervention. The objective of this application, which is a step in pursuit of that goal, is to define the molecular mechanisms responsible for Vpr's activity in HIV-1 nuclear importation. The central hypothesis of the application is that Vpr (or Hsp70) critically regulates translocation and docking of the HIV-1 preintegration complex to the nuclear envelope, but then dissociates from the preintegration complex to allow its migration through the pore and to the integration site. This hypothesis reconciles findings from our laboratory and reports published by other groups. The rationale for the proposed research is that, once the mechanisms of Vpr/Hsp70 activity are known, agents can be designed to inhibit HIV-1 nuclear import and thus abort infection at a pre-integration step. The central hypothesis will be tested and the objective of the application accomplished by pursuing four specific aims: 1) Characterize HIV-1 preintegration complexes at various stages of maturation; 2) Determine at which step of HIV-1 nuclear import Vpr exerts its activity; 3) Characterize the mechanism(s) of Vpr's nuclear import activity; and 4) Investigate the mechanism of activity of Hsp70 in (HIV-1) nuclear import and its relation to Vpr. The proposed work is innovative, as it capitalizes on recent findings in the field of HIV-1 nuclear import, and will provide important, new information on the previously unknown events and mechanisms governing nuclear translocation of the HIV-1 genome. These results will be significant, because they are expected to facilitate discovery of agents that would inhibit Vpr activity and thus attenuate HIV replication.